Phosphorylation of cPLA2α at Ser505 Is Necessary for Its Translocation to PtdInsP2-Enriched Membranes

Group IVA cytosolic phospholipase A2α (cPLA2α) is a key enzyme in physiology and pathophysiology because it constitutes a rate-limiting step in the pathway for the generation of pro- and anti-inflammatory eicosanoid lipid mediators. cPLA2α activity is tightly regulated by multiple factors, including the intracellular Ca2+ concentration, phosphorylation reactions, and cellular phosphatidylinositol (4,5) bisphosphate levels (PtdInsP2). In the present work, we demonstrate that phosphorylation of the enzyme at Ser505 is an important step for the translocation of the enzyme to PtdInsP2–enriched membranes in human cells. Constructs of eGFP-cPLA2 mutated in Ser505 to Ala (S505A) exhibit a delayed translocation in response to elevated intracellular Ca2+, and also in response to increases in intracellular PtdInsP2 levels. Conversely, translocation of a phosphorylation mimic mutant (S505E) is fully observed in response to cellular increases in PtdInsP2 levels. Collectively, these results suggest that phosphorylation of cPLA2α at Ser505 is necessary for the enzyme to translocate to internal membranes and mobilize arachidonic acid for eicosanoid synthesis.


Introduction
The eicosanoids constitute an ample family of bioactive lipids with potent pro-and anti-inflammatory activities. They are not stored in the cells but produced in response to specific cellular stimulation [1,2]. The common precursor of the eicosanoids is arachidonic acid (AA), a fatty acid that is mostly found esterified in the sn-2 position of cellular glycerophospholipids, primarily those containing choline, ethanolamine, and inositol as polar headgroups [3][4][5]. Although the cells possess multiple phospholipase A 2 (PLA 2 ) enzymes potentially capable of liberating AA from membrane phospholipids [6][7][8][9], the major PLA 2 involved in receptor-mediated AA mobilization is the group IVA PLA 2 , also known as cytosolic PLA 2 α (cPLA 2 α) [10,11]. cPLA 2 α is tightly regulated in cells, not only to finely regulate the amount of AA mobilized for eicosanoid synthesis but also because both of its by-products, free fatty acids and lysophospholipids, could be deleterious to the cells if accumulating at high levels. Increases in the intracellular Ca 2+ concentration constitute one of the key regulators of cPLA 2 α activity in cells and mediate translocation of the enzyme to a variety of cytoplasmic membranes [10,11]. This is driven by the presence in the enzyme of a calcium-binding domain, or C2 domain. Unlike other PLA 2 family members, cPLA 2 α does not require Ca 2+ for enzyme activity, but to dock and penetrate into the membrane interface [12][13][14].
In addition to Ca 2+ , cPLA 2 α is also regulated by intracellular lipids. The C2 domain also has a site for ceramide 1-phosphate, produced by activated ceramide kinase in situ [15,16]. This lipid allosterically activates the enzyme and increases the residence  [15,16]. cPLA 2 α also binds phosphatidylinositol 4,5bisphosphate (PtdInsP 2 ) via a 4-Lys cluster present in the catalytic domain [17,18]. In vitro, PtdInsP 2 increases the catalytic activity of the enzyme in a calcium-independent manner, likely by enhancing its capacity to penetrate membranes, especially those enriched in choline phospholipids [17,18]. Mutations in the residues where PtdInsP 2 binds give rise to an enzyme that, when transfected into cells, manifests a reduced ability to translocate to intracellular membranes and mobilize AA [19,20]. cPLA 2 α can also be phosphorylated in cells at residues Ser 505 , Ser 515 , and Ser 727 , and all of these phosphorylation reactions have been suggested to be involved in the regulation of agonist-induced AA mobilization [10,11,21]. While the functional relevance of cPLA 2 α phosphorylation at Ser 515 and Ser 727 may depend on cell type and stimulation conditions, there is general agreement that phosphorylation at Ser 505 represents a key regulatory event under nearly all cellular conditions examined; thus, it has been the most extensively studied [10,11,21]. In general terms, it appears that the extracellularregulated kinases p42/p44 are responsible for cPLA 2 α phosphorylation at Ser 505 in cells of murine origin [22][23][24], and the related kinases p38 and JNK are involved in cells of human origin [25][26][27].
Several lines of evidence have suggested that cPLA 2 α phosphorylation at Ser 505 is necessary for the enzyme to be fully active in cells; however, the molecular reasons for this still remain elusive [21]. In vitro studies have indicated that phosphorylation of cPLA 2 α at Ser 505 is not required for activity but for proper binding of the enzyme to membranes in a Ca 2+ -dependent fashion [28].
Previous work from our laboratory has highlighted the importance of intracellular PtdInsP 2 levels in regulating the physical state of cPLA 2 α at Ca 2+ levels equaling those present in resting cells [19]. The phosphorylation state of cPLA 2 α does not seem to influence the translocation of the enzyme to model membranes in the presence of high Ca 2+ [28]. In the present study, we have studied the influence of cPLA 2 α phosphorylation at Ser 505 on PtdInsP 2 binding and in the translocation capacity of the enzyme in a cellular scenario. We show that cPLA 2 α phosphorylation at Ser 505 is necessary for the translocation of the enzyme to membranes and to promote AA release in response to PtdInsP 2 elevations. These studies provide new insights into the complex regulation of cPLA 2 α, thereby expanding and deepening our knowledge of the cellular mechanisms controlling the production of pro-and anti-inflammatory lipid mediators.

Role of cPLA 2 α Phosphorylation in Membrane Translocation in Response to Increases in PtdInsP 2 and Calcium
In previous work, we showed that increasing PtdInsP 2 levels in intact cells in the absence of a rise in intracellular Ca 2+ is sufficient to trigger cPLA 2 α activation and attendant AA mobilization [19]. We began the current study by assessing whether such an effect of PtdInsP 2 requires the enzyme to be phosphorylated at Ser 505 . To this end, cells stably expressing eGFP-cPLA 2 α or the mutant eCFP-S505A-cPLA 2 α were incubated with exogenous PtdsInsP 2 complexed with a histone shuttle to facilitate entry into the cells. We and others have previously used this technique successfully [19,29,30], which results in cells displaying increased PtdInsP 2 levels, and no influence on the physical state of cPLA 2 α, as the incubations are carried out in the absence of extracellular Ca 2+ [19]. The PtdsInsP 2 contained a fluorescent tag that allowed its monitoring within the cells by confocal microscopy. Once the cells incorporated all the PtdsInsP 2 (as assessed by the cellular fluorescence associated with it, which remained stable and did not increase further), restoring extracellular Ca 2+ levels triggered the immediate translocation of cPLA 2 α, i.e., 1 min, to internal membranes ( Figure 1A). Importantly, the non-phosphorylatable eCFP-S505A-cPLA 2 α mutant required considerably more time, i.e., 5-12 min, to translocate to perinuclear membranes ( Figure 1B). Because PtdInsP2 is known to reduce the Ca 2+ threshold for cPLA2α to translocate t membranes in vitro [17,31], in the next series of experiments, we examined the behavior o the enzyme and the S505A mutant under the opposite circumstances, i.e., in the presence o a sustained rise of the intracellular Ca 2+ level. As shown in Figure 2, cell treatment with µM ionomycin, which raises the intracellular Ca 2+ concentration up to 4 µM [19,32], th eCFP-S505A-cPLA2α translocated to inner membranes after experiencing a marked dela compared with wild type EGFP-cPLA2α. The former required approx. 10 min to target th perinuclear membranes ( Figure 2B), while the latter was translocated completely to per nuclear membranes within the first 3 min of treatment ( Figure 2A). Collectively, these dat indicate that phosphorylation of cPLA2α at Ser 505 is a key step for the enzyme to readil translocate to its target membranes even at high intracellular Ca 2+ levels. Because PtdInsP 2 is known to reduce the Ca 2+ threshold for cPLA 2 α to translocate to membranes in vitro [17,31], in the next series of experiments, we examined the behavior of the enzyme and the S505A mutant under the opposite circumstances, i.e., in the presence of a sustained rise of the intracellular Ca 2+ level. As shown in Figure 2, cell treatment with 5 µM ionomycin, which raises the intracellular Ca 2+ concentration up to 4 µM [19,32], the eCFP-S505A-cPLA 2 α translocated to inner membranes after experiencing a marked delay compared with wild type EGFP-cPLA 2 α. The former required approx. 10 min to target the perinuclear membranes ( Figure 2B), while the latter was translocated completely to perinuclear membranes within the first 3 min of treatment ( Figure 2A). Collectively, these data indicate that phosphorylation of cPLA 2 α at Ser 505 is a key step for the enzyme to readily translocate to its target membranes even at high intracellular Ca 2+ levels. Because PtdInsP2 is known to reduce the Ca 2+ threshold for cPLA2α to translocate t membranes in vitro [17,31], in the next series of experiments, we examined the behavior o the enzyme and the S505A mutant under the opposite circumstances, i.e., in the presence o a sustained rise of the intracellular Ca 2+ level. As shown in Figure 2, cell treatment with µM ionomycin, which raises the intracellular Ca 2+ concentration up to 4 µM [19,32], th eCFP-S505A-cPLA2α translocated to inner membranes after experiencing a marked dela compared with wild type EGFP-cPLA2α. The former required approx. 10 min to target th perinuclear membranes ( Figure 2B), while the latter was translocated completely to per nuclear membranes within the first 3 min of treatment ( Figure 2A). Collectively, these dat indicate that phosphorylation of cPLA2α at Ser 505 is a key step for the enzyme to readil translocate to its target membranes even at high intracellular Ca 2+ levels. To assess the possible physiological/pathophysiological relevance of the delay in membrane translocation of the eCFP-S505A-cPLA 2 α, we also conducted AA mobilization experiments under identical experimental conditions. The cells were prelabeled with [ 3 H]AA, and the release of radiolabeled fatty acid was measured after exposing the cells to PtdInsP 2 and ionomycin. As shown in Figure 3, [ 3 H]AA release in cells transfected with the eCFP-S505A-cPLA 2 α mutant was significantly lower than that of cells transfected with the wild-type enzyme, and this occurred at all conditions tested. Thus, delayed cPLA 2 α translocation results in diminished fatty acid mobilization.

Figure 2.
Translocation of eGFP-cPLA2α and eCFP-S505A-cPLA2α in response to high Ca 2+ concentrations. Fluorescence from HEK cells stably transfected with eGFP-cPLA2α (A) or the mutant eCFP-S505A-cPLA2α (B) was analyzed by confocal microscopy before (Control) or after stimulation with 5 µM ionomycin for the indicated periods of time.

Phosphorylation of cPLA2α Is Necessary for AA Release in Response to PtdInsP2
To assess the possible physiological/pathophysiological relevance of the delay in membrane translocation of the eCFP-S505A-cPLA2α, we also conducted AA mobilization experiments under identical experimental conditions. The cells were prelabeled with [ 3 H]AA, and the release of radiolabeled fatty acid was measured after exposing the cells to PtdInsP2 and ionomycin. As shown in Figure 3, [ 3 H]AA release in cells transfected with the eCFP-S505A-cPLA2α mutant was significantly lower than that of cells transfected with the wild-type enzyme, and this occurred at all conditions tested. Thus, delayed cPLA2α translocation results in diminished fatty acid mobilization. To further characterize the importance of Ser 505 phosphorylation in cPLA2α translocation in response to PtdInsP2, mutants were constructed where Ser 505 was replaced with Glu (S505E), which mimics phosphorylation at that residue [25,28]. The ability of this S505E mutant to translocate in response to PtdInsP2 elevations and to sustained increases in intracellular Ca 2+ was then evaluated, and the results are shown in Figure 4. As expected, the S505E mutant behaved the same as the wild-type mutant ( Figure 4A-D). Note that, in common with many other cells in culture [24,33], most of the wild-type cPLA2α in the HEK cells is already phosphorylated at Ser 505 . This explains why the S505E mutant behaves the same as the wild-type enzyme. Moreover, cells expressing the S505E mutant manifested an AA release response to either PtdInsP2 elevations or ionomycin which was essentially identical to that of cells expressing the wild-type enzyme ( Figure 4E). Thus, these data show that cPLA2α phosphorylation at Ser 505 capacitates the enzyme for a full functional response. To further characterize the importance of Ser 505 phosphorylation in cPLA 2 α translocation in response to PtdInsP 2 , mutants were constructed where Ser 505 was replaced with Glu (S505E), which mimics phosphorylation at that residue [25,28]. The ability of this S505E mutant to translocate in response to PtdInsP 2 elevations and to sustained increases in intracellular Ca 2+ was then evaluated, and the results are shown in Figure 4. As expected, the S505E mutant behaved the same as the wild-type mutant ( Figure 4A-D). Note that, in common with many other cells in culture [24,33], most of the wild-type cPLA 2 α in the HEK cells is already phosphorylated at Ser 505 . This explains why the S505E mutant behaves the same as the wild-type enzyme. Moreover, cells expressing the S505E mutant manifested an AA release response to either PtdInsP 2 elevations or ionomycin which was essentially identical to that of cells expressing the wild-type enzyme ( Figure 4E). Thus, these data show that cPLA 2 α phosphorylation at Ser 505 capacitates the enzyme for a full functional response.

Discussion
The mechanisms responsible for the translocation of cPLA 2 α to cellular membranes in the absence of sustained increases in intracellular calcium have remained a subject of debate [10,11,34]. Early studies utilizing purified cPLA 2 α showed that the binding of the enzyme to vesicles and micelles increased in the presence of PtdInsP 2 , resulting in enhanced activity even at nanomolar Ca 2+ levels [17,18,31]. Studies in intact cells have also provided evidence that increased PtdInsP 2 levels in cells can sustain cPLA 2 α activation and attendant AA mobilization at Ca 2+ levels equaling those of resting cells [34,35]. Finally, a four-Lys cluster was described in the enzyme, which binds PtdInsP 2 tightly and may help regulate the cellular location of the enzyme under stimulatory conditions [19,20]. In this work, we extend our knowledge of PtdInsP 2 regulation of cPLA 2 α by showing for the first time that phosphorylation of the enzyme at Ser 505 is necessary for the full regulatory effect of PtdInsP 2 to take place. Thus, these results establish a hitherto unrecognized link between two major mechanisms of cPLA 2 α regulation, namely PtdInsP 2 and Ser 505 phosphorylation.
While phosphorylation of cPLA 2 α at Ser 505 has been recognized for a long time, its full physiological significance remains unclear. Reasons for this include the finding that in resting cells, most of the cPLA 2 α is already phosphorylated at Ser 505 and that the specific activity of the non-phosphorylated enzyme differs little from that of the phosphorylated one [24,33,36]. Our data support the view that PtdInsP 2 may help the cPLA 2 α to achieve the appropriate conformation for optimal interaction of the enzyme with its target membrane, in agreement with previous observations [28]. cPLA 2 α is a rather 'promiscuous' enzyme, being able to translocate to different membranes depending on cell type and stimulatory conditions [11,37]. Whether PtdInsP 2 regulates the translocation of cPLA 2 α to all kinds of intracellular membranes or its regulatory function is limited to targeting the enzyme to specific membranes is unknown at present. It is also interesting to note that the kinases involved in phosphorylating cPLA 2 α at Ser 505 under activation conditions appear to greatly depend on cell type and stimulation conditions [22][23][24][25][26][27]. Whether these differences are due to species-specific features or reflect distinctive regulatory attributes of the enzyme is also unknown. Future work in the laboratory will be aimed to investigate whether the PtdInsP 2 effects on enzyme translocation are related to the involvement of a specific kinase or intracellular membrane.
Stimulatory cell conditions of physiological/pathophysiological relevance do not lead to high calcium concentrations inside the cell. Rather, receptor-mediated activation promotes low and transient increases in Ca 2+ concentration, which on many occasions lead to cPLA 2 α activation [10][11][12]. Thus, it seems necessary to define the factors that regulate the translocation of cPLA 2 α to membranes under physiological Ca 2+ conditions. Several lines of evidence have suggested that the enzyme behaves differently depending on Ca 2+ availability. Under high Ca 2+ concentrations (>1 µM), the C2 domain of the enzyme is fully active and can drive translocation of the cPLA 2 α to membranes without any other requirement [12,34]. On the contrary, under physiologically relevant Ca 2+ conditions (up to 400 nM), multiple regulatory components may have to be set into motion to achieve full translocation of the enzyme. We have shown here that cPLA 2 α phosphorylation at Ser 505 is one of these components.
The finding that the non-phosphorylatable mutant S505A shows a reduced ability to translocate to membranes in response to PtdInsP 2 elevations suggests that, in the absence of phosphorylation, cPLA 2 α is not capable of binding productively to PtdInsP 2 , probably because the affinity for the phospholipid is decreased. Alternatively, it is also possible that the S505A mutant requires higher Ca 2+ levels to translocate to the membrane, even in the presence of PtdInsP 2 . The experiments conducted with the phosphorylation mimetic mutant S505E further support the idea that cPLA 2 α phosphorylation at Ser 505 is required for the enzyme to recognize and respond to PtdInsP 2 elevations optimally.
Overall, the findings described here demonstrate that cPLA 2 α has multiple mechanisms to circumvent its necessity for high Ca 2+ concentrations to translocate to membranes and that those mechanisms interact with each other. Moreover, the diminished translocation ability of the nonphosphorylated enzyme in response to PtdInsP 2 elevations (at least 11 min delay compared with the wild-type enzyme) underscores the importance of these interactions for cPLA 2 α to display full biological activity.

Lipid Preparation
PtdInsP 2 was added to the cells as previously described [19,30]. Briefly, 2 µg of phospholipid was mixed with 2 µL of the carrier (histone, 0.5 mM), resuspended in Hank's balanced salt solution containing 10 mM HEPES, sonicated in a water bath for 2 min, and allowed to rest at 37 • C for 10 min before use. The final concentration of PtdInsP 2 in the incubation media was 5.7 µM.

Confocal Microscopy
The cells were seeded on glass-bottom culture dishes (MatTek Corp., Ashland, MA, USA) and allowed to adhere for 24 h. The medium was then replaced by Hanks' buffered saline containing 10 mM HEPES and 1.3 mM CaCl 2 . For some experiments, cells were incubated without CaCl 2 , which was added back when needed. Fluorescence was monitored by confocal microscopy using a Bio-Rad Radiance 2100 laser-scanning system coupled to a Nikon TE-2000U with a termostatized chamber (Warner Instruments, Holliston, MA, USA). The objective was CFI Plan Apo 60X, 1.4 numerical aperture, and oil immersion. The fluorescence of eCFP was monitored at 457 nm argon excitation using the combination of a long pass barrier filter HQ470LP and a short pass filter HQ520SP. The fluorescence of eGFP was monitored at 488 nm Argon excitation using the combination of a long pass filter HQ500LP and a short pass filter HQ560SP. The Alexa-Fluo 594 fluorescence was monitored at 543 nm HeNe excitation using a long band pass filter HQ570LP. Red fluorescence from BODYPI-TRx was monitored at 543 nm HeNe laser excitation using an HQ590/570 long band pass blocking filter.

AA Release
The cells were labeled with 0.5 µCi/mL [ 3 H]AA (sp. act. 200 Ci/mmol; HartBiomédica, Madrid, Spain) for 18 h. Afterward, they were washed extensively and overlaid with 0.5 mL of serum-free medium supplemented with 0.5 mg/mL albumin and treated with 5 µM thimerosal for 15 min to block fatty acid reacylation [38,39]. The cells were then stimulated for 60 min. Supernatants were removed, and cell monolayers were overlaid with ice-cold phosphate buffer containing 0.05% Triton X-100 and scraped. Total lipids from supernatants and cells were extracted according to Bligh and Dyer [40]. After extraction, lipids were separated by thin-layer chromatography using the system n-hexane/diethyl ether/acetic acid (70:30:1 by volume) [41]. Spots corresponding to free AA and phospholipid were scraped, and radioactivity was quantified by liquid scintillation counting.

Conclusions
cPLA 2 α-mediated production of bioactive lipid mediators represents a key event in the execution of physiological and pathophysiological responses to external stimuli. This study has focused on the complex interactions that govern cPLA 2 α translocation to membranes and the multiple factors that may be involved in its regulation. Specifically, we have described the interconnected role of two of these factors, phosphorylation of the enzyme at Ser 505 and cellular PtdInsP 2 levels. Both of them seem to work together to promote membrane translocation and activation of cPLA 2 α under low intracellular Ca 2+ levels. Together, the studies described here represent a relevant working model to further understand the intricacies of the cellular regulation of cPLA 2 α and the molecular mechanisms underlying it. Funding: This research was funded by the Spanish Ministry of Science and Innovation (MICIN/AEI/ 10.13039/501100011033), grant number PID2019-105989RB-I00, and the the Regional Government of Castile and Leon, grant number CSI141P20 co-financed by the European Union through the European Regional Development Fund (ERDF A Way of Making Europe). The APC was funded by the Spanish National Research Council (CSIC). CIBERDEM is an initiative of Instituto de Salud Carlos III.

Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained within the article.